The long term goal of this project is to provide an experimental basis for using gene therapy to reconstitute the immune function of patients with HIV infection. This is to be accomplished by protecting the progeny of the hematopoietic stem cells and rendering them resistant to HIV infection through transfer of the anti-HIV ribozyme genes into pluripotent stem cells and CD34+ subsets. The immediate objectives are: (1) To optimize the conditions of transduction of anti-HlV ribozyme gene constructs into human CD34 + cells from different sources, i.e., bone marrow, fetal cord blood, and blood stem cells; (2) To assess and improve the stability and expression of the transgenes in the CD34+ subsets and their progeny cells after transduction and culture in short and long-term cultures; (3) To evaluate the protective effect of the.ribozyme gene products against the challenge of HIV exposure in the hematopoietic progeny cells. (4) To explore the optimal conditions for ex vivo expansion of the transduced CD34+ cells. The knowledge gained from this study will be translated into implementation of gene therapy for patients with AIDS. Eventually autologous or allogeneic transplantation of the genetically manipulated stem cells will represent an ultimate cure. To achieve these goals, the following experimental design and methods are used: (1) Enrichment of the stem cells defined as CD34 + cells from bone marrow, fetal cord, and placental blood, as well as from peripheral blood by means of an immunomagnetic microsphere method, initially in research scale and subsequently in clinical scale; (2) Establishment of optimal conditions for the delivery of anti-HlV ribozyme genes into the CD34 + cells and their subsets: stimulation with (or without) cytokines, manipulation of micro environment, serum free media, optimization of the vectors for anti- HlV ribozyme genes. (3) Assessment of stability and expression of the transgenes by reverse transcriptase-polymerase chain reaction in short and long-term cultures of the stem cells; (4) Evaluation of the protective function of the ribozyme genes by challenging the mononuclear progeny cells with HIV virus; (5) Establishment of optimal conditions for expansion of transducted CD34+ cells. The research group of the P.I. focuses on stem cell research and bone marrow transplantation. The component on CD34+ cell isolation, culture and expansion will be performed by the group of Dr. Ho and the preparation of anti-HIV-ribozyme genes, production of the vectors and challenging with HIV virus will be performed by Dr. Flossie Wong-Staal's group.